The goal of this proposal is development of a multiplexed, portable real time evanescent fiber optic sensor instrument for quantifying the amount of specific DNA in a sample. During Phase I of this project, IA demonstrated the feasibility of monitoring DNA hybridization in real time using an evanescent fiber-optic DNA biosensor with fluorescence detection. The DNA biosensor was mounted within a temperature controlled reaction chamber. DNA sequences were amplified using the polymerase chain reaction (PCR) and hybridized to specific probes immobilized to the DNA biosensor surface. During Phase II, IA will develop a multiplexed DNA Biosensor Fluorometer capable of simultaneously monitoring the hybridization occurring on eight or more DNA biosensors at a time. The increased sensitivity associated with evanescent sensing will allow detection of one DNA copy after 25-30 cycles. The small size of the instrument and associated sample prep cartridge will adapt it to point of care testing for pathogen identification. Specific Aim 1 provides for incorporation into the instrumental methods, use of a probe for an internal PCR standard for normalization of the efficiency of each individual reaction. Specific Aim 2 provides a multiplexed sensor cartridge so that each cartridge can run eight samples simultaneously. Specific Aim 3 provides comparison of performance of the prototype instrument with established methods using plasmids containing sequences which are specific for HPV 16 or for Chiamydia trachomatis. A specialized cartridge for sample preparation will be developed under Specific Aim 4. This will allow biological samples of clinical interest to be prepared for PCR without the need of a centrifuge. Sample will be applied to the top of the cartridge and reagents will be pressed through with a syringe. DNA will be eluted ready for amplification in the easily portable sensor instrument. PCR will be performed in situ in the sensor cartridge while the instrument records the rate of hybridization for eight solutions and standards. This will provide for a control sample and a standard curve over an anticipated seven log range. The system can be adapted to a variety of amplification formats, and has the potential for use in genetics research laboratories and for clinical determinations in hospital labs. It will provide an instrument and sample preparation method uniquely adapted to point of care testing for pathogens in a short period of time.